1Department of Pediatrics, University of Rochester, Rochester, New York 14642, USA.

Abstract

Our previous studies demonstrated that p75NTR confers protection against oxidative stress-induced apoptosis upon PC12 cells; however, the mechanisms responsible for this effect are not known. The present studies reveal decreased mitochondrion membrane potential and increased generation of reactive oxygen species (ROS) in p75NTR-deficient PC12 cells as well as diminution of ROS generation after transfection of a full-length p75NTR construct into these cells. They also show that p75NTR deficiency attenuates activation of the phosphatidylinositol 3-kinase --> phospho-Akt/protein kinase B pathway in PC12 cells by oxidative stress or neurotrophic ligands and inhibition of Akt phosphorylation decreases the glutathione (GSH) content in PC12 cells. In addition, decreased de novo GSH synthesis and increased GSH consumption are observed in p75NTR-deficient cells. These findings indicate that p75NTR regulates cellular handling of ROS to effect a survival response to oxidative stress.

(a) Generation of peroxide in p75NTR-deficient (left panels) and native PC12 cells (right panels) under basal conditions (vehicle) and after 6-OHDA treatment (6-OHDA; 40μg/mL, 24 h) as determined with carboxy-H2DCFDA. Bright field images indicate the total number of cells in the field corresponding to the carboxy-H2DCFDA stained images above them. Under both conditions, the number of fluorescent cells is much greater in p75NTR-deficient cells. (b) Quantification of carboxy-H2DCFDA fluorescence using a microplate reader. The number of viable cells in each well was determined by Alamar blue assay and carboxy-H2DCFDA fluorescence was corrected for Alamar blue absorbance. The intensity of fluorescence is significantly higher in p75NTR-deficient cells with or without 6-OHDA treatment. The level of fluorescence increased with 20μg/mL 6-OHDA in p75NTR-deficient cells while in native cells the difference was detected only at 40μg/mL. (c) Transfection of a full-length p75NTR construct into p75NTR-deficient PC12 cells reduced the number of carboxy-H2DCFDA positive cells both under basal conditions (vehicle) and after 6-OHDA (40 μg/ mL, 24 h) treatment (6-OHDA). Bright field images indicate the total number of cells in the field corresponding to the carboxy-H2DCFDA stained images above them. (d) Quantification of carboxy-H2DCFDA fluorescence in mock- and p75NTR-transfected p75NTR-deficient PC12 cells using a microplate reader. The intensity of the fluorescence is significantly higher in mock-transfected p75NTR-deficient cells both under basal conditions and after 6-OHDA treatment. The level of fluorescence increased with 20μg/mL 6-OHDA in mock-transfected cells while in p75NTR-transfected cells the difference was detected only at 40 μg/mL. Mean±SE; *p < 0.05; ***p < 0.001; n = 4; two-way ANOVA and the Bonferroni post hoc test.

(a) Generation of cellular superoxide in p75NTR-deficient (left panels) and native PC12 cells (right panels) under basal conditions (top) and after 6-OHDA treatment (20 μg/mL; middle and 40 μg/mL; bottom) as detected with DHE. Under resting conditions, the intensity of the fluorescence in p75NTR-deficient cells is much weaker (top left) compared to that in native PC12 cells (top right). The level of fluorescence after 20 μg/mL 6-OHDA treatment is comparable between the two cell lines (middle). After 40 μg/mL 6-OHDA treatment, the majority of the nuclei in p75NTR-deficient cells appear fluorescent (a sign of cell damage and binding of DHE to nucleic acid; bottom left) while only a few such nuclei are found in native PC12 cells (bottom right). (b) Quantification of DHE fluorescence using a microplate reader. The number of viable cells in each well was determined by Alamar blue assay and DHE fluorescence was corrected for Alamar blue absorbance. Although the intensity of the fluorescence is significantly lower in p75NTR-deficient cells as compared to the native PC12 cells under basal conditions, the level of fluorescence is much higher in p75NTR-deficient cells after 40 μg/mL 6-OHDA treatment. In addition while the staining in p75NTR-deficient cells after 10–20 μg/ mL 6-OHDA treatment is significantly higher than under control conditions, the staining in native PC12 cells appeared comparable to that seen under control conditions and after 10–20 μg/mL 6-OHDA treatment. (c) Staining with MitoSOX Red superoxide indicator shows the generation of mitochondrial superoxide in p75NTR-deficient (left panels) and native PC12 cells (right panels) under basal conditions (top) and after 6-OHDA (10 μg/mL; middle and 40 μg/mL; bottom; 24 h) treatment. Although p75NTR-deficient cells exhibited more severe damage (redistribution of fluorescence to nuclei and the cytosol; bottom left) than native cells (minor mitochondrial disruption; bottom right) after 40 μg/mL 6-OHDA treatment, the level of mitochondrial superoxide generation appeared comparable in the two cell lines under resting conditions (top) and after 10 μg/mL 6-OHDA treatment (middle). (d) Quantification of MitoSOX Red fluorescence using a microplate reader. The number of viable cells in each well was determined by Alamar blue assay and MitoSOX Red was corrected for Alamar blue absorbance. The intensity of the fluorescence in both cell lines is comparable under basal conditions and after 10–20 μg/mL 6-OHDA treatment. The level of fluorescence is significantly higher in p75NTR-deficient cells after 40 μg/mL 6-OHDA treatment as compared with that seen in native PC12 cells. (e) Mitochondrial membrane potential (Δψm) in p75NTR-deficient and native PC12 cells under basal conditions and after 6-OHDA treatment as determined with JC-1 staining. The Δψm indicated by the ratio of J-aggregates and monomers is significantly lower in p75NTR-deficient cells with or without 6-OHDA treatment. The level of the Δψm decreased with 20 μg/mL 6-OHDA in p75NTR-deficient cells while in native cells the difference was detected only at 40 μg/mL. Mean ± SE; *p < 0.05; **p < 0.01; n = 4; two-way ANOVA and the Bonferroni post hoc test.

(a and b) Phosphorylated Akt is reduced in p75NTR-deficient PC12 cells. (a) p75NTR-deficient (−) and Native (+) and PC12 cells were maintained in low serum medium (DMEM containing 0.5% FBS) for 16 h prior to being incubated in medium containing 10% HS and 5% FBS (serum, 24 h), low serum medium (lowSerum, 24 h), and low serum medium followed by 100 ng/mL NGF treatment for 1 h (NGF 1 h) and 24 h (NGF 24 h). Cells were harvested and prepared for western blot analysis using anti-phospho-Akt (p-Akt) and anti-Akt (Akt) antibodies. β-actin is included for loading control. (b) Densitometric analysis of phospho-Akt normalized to total Akt. In native PC12 cells, approximately 10% of total Akt is phosphorylated when maintained in serum-containing medium and the level of phosphorylation increased to almost 45% when examined at 1 h after NGF treatment. In p75NTR-deficient cells, Akt phosphorylation is barely detectible in serum-containing medium and the level of phosphorylation is slightly increased at 1 h after NGF treatment, but the degree of increase is far less than that in native PC12 cells. Phosphorylated Akt is undetectable in both types of cells when starved overnight. (c and d) Western blot analysis showing that the increase in activation of Akt after H2O2 treatment in p75NTR-deficient cells is attenuated as compared with the native PC12 cells. (c) p75NTR-deficient (−) and native PC12 (+) cells were treated with 0.5 mM H2O2 for the indicated time. At the end of each time period cells were collected and prepared for western blot analysis using anti-phospho-Akt (p-Akt) and anti-Akt (Akt) antibodies. While the level of Akt phosphorylation increased in both types of cells following H2O2 treatment, the increase in native PC12 cells was much greater and more prolonged than that in p75NTR-deficient cells PC12 cells. (d) Densitometric analysis of phospho-Akt normalized to total Akt. Mean±SE; *p < 0.05; **p < 0.01; n = 3; two-way ANOVA and the Bonferroni post hoc test.

(a–d) Western blot analysis showing the activation of Akt in p75NTR-deficient and native PC12 cells (a and b) as well as in p75NTR-deficient cells that had been transfected with either a plasmid containing the full-length p75NTR construct (p75NTR-overEx) or a mock plasmid (mock) after 6-OHDA treatment (c and d). Cells were treated with vehicle or 40 μg/mL 6-OHDA for 1 h and prepared for western blot analysis using anti-phospho-Akt (p-Akt) and anti-Akt (Akt) antibodies. (a) The level of Akt phosphorylation in native and p75NTR-deficient PC12 cells in both control and 6-OHDA-treated conditions. (b) Densitometric analysis of phospho-Akt normalized to total Akt in (a). (c) Akt phosphorylation in cells transfected with mock or p75NTR constructs in both control and 6-OHDA-treated conditions. (d) Densitometric analysis of phospho-Akt normalized to total Akt in (c). (e–g) Inhibition of PI3 kinase decreased GSH contents in native and p75NTR-defi-cient PC12 cells. Cells were treated with LY294002 for 16 h at the indicated concentrations. Inhibition of Akt phosphorylation is confirmed by western blot analysis (e and f) and cellular GSH was measured using ThioGlo-1(g). (e) Western blot analysis showing the reduced level of Akt phosphorylation after 10 and 30 μM LY294002 treatment. (f) Densitometric analysis of phospho-Akt normalized to total Akt. (g) GSH contents in native and p75NTR-deficient PC12 cells measured after vehicle and LY294002 treatment. Mean±SE; *p < 0.05; **p < 0.01; n = 3 in (f); n = 10 in (g); one-way ANOVA and the Bonferroni post hoc test.